Hrp conjugated goat anti mouse igm

Serum was collected from individual naïve chimera mice four to five weeks post transplantation or from 3-month old BALB/c-ByJ mice at the time of euthanasia. The serum was analyzed for total IgM by ELISA according to the manufacturer’s instructions (Bethyl Laboratories). IgM specific PC and PPS3 serum levels were measured by coating 96-well plates with PC-BSA (Biosearch Technologies) or PPS3 (American Type Culture Collection) at 5 μg/ml in 1X PBS. All plates were washed with TBS Tween (0.05%) and blocked with 1% BSA for 1 hour at room temperature. Bound IgM was measured using HRP-conjugated goat anti-mouse IgM (Bethyl Labs). IgM standards were included on each plate and PC / PPS3 specific antibody levels were interpreted as volume equivalent of IgM, as described in (31 (link)).

Serum was collected from individual BALB/c-ByJ naïve mice at the time of euthanasia at the ages indicated. The serum was analyzed for antibody against DMPC and DOPC by ELISA. ELISA strips were obtained from Avanti Polar Lipids pre-coated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The wells were blocked with 200μl of 3% fatty acid free bovine serum albumin in PBS for one hour at room temperature with gentle shaking. The wells were then washed three times with 1X PBS. Diluted serum was added at 50μl per well and incubated for one hour at room temperature with gentle shaking. The wells were then washed three times with 1X PBS. Bound antibody was measured using HRP-conjugated goat anti-mouse IgM (Bethyl Labs) at 1:20,000. NC-17, kindly provided by Dr. Gregg Silverman, was used as a standard and included on each plate.